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Abstract

Reactive oxygen species (ROS) modulate both physiological and pathophysiological responses in the cardiovascular system. Increased ROS production has been implicated in the pathogenesis of cardiovascular diseases. Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in many cell types. Caveolin-1null mice are viable but have marked cardiovascular phenotypes, including pulmonary arterial hypertension and cardiac hypertrophy. However, the molecular mechanisms whereby loss of caveolin-1 leads to these abnormalities are incompletely understood. We hypothesized that caveolin-1 regulates cellular and organismal redox state in these cardiovascular tissues. We found that plasma 8-isoprostanes levels were strikingly higher in caveolin-1null mice than wild-type mice (0.2 ± 0.1 vs. 9.0 ± 0.5 pg/ml), indicating that caveolin-1null mice have markedly increased oxidative stress. To explore the underlying mechanisms, we exploited small interfering RNA (siRNA) approaches to selectively knock down caveolin-1 and related signaling proteins in cultured endothelial cells. We found that siRNA-mediated caveolin-1 knockdown resulted in a significant 42±3 % increase (n=6, P<0.01) in extracellular hydrogen peroxide (H2O2) levels, quantitated using the Amplex Red assay. Simultaneous siRNA-mediated knockdown of AMP-activated kinase, Rac1 or eNOS did not affect the increase in H2O2 levels seen following caveolin-1 knockdown in these cells. Pharmacological inhibitors of mitochondrial respiration (rotenone) or NADPH oxidases (VAS2870) failed to attenuate the increase in H2O2 levels seen following caveolin-1 knockdown. We next transfected endothelial cells with the H2O2 biosensor HyPer2, and found no change in intracellular H2O2 concentrations following caveolin-1 knockdown. These results taken together indicate that caveolin-1 may regulate membrane transport of H2O2 in endothelial cells. These findings establish that caveolin-1 plays a key role in the regulation of cellular and organismal oxidative stress, and suggest that caveolin-1-modulated pathways may represent novel targets for the amelioration of oxidative stress in cardiovascular diseases.